A mixture of compounds, or analytes, can be separated by pumping the mixture through a separating device such as a chromatographic column using a process known as liquid chromatography, a variant of which is known as high performance liquid chromatography (HPLC). The separation of the sample is caused by analytes having different affinity for the chromatographic packing material within the column. The separated sample flows out of the chromatographic column continuously, but with the separated analytes emerging from the column at different times. The individual compounds comprising the analyte may then pass through various detection devices such as an ultraviolet light absorbance detector, a mass spectrometer, a fluorescence detector and the like to assist in determining the composition of the sample. The analytes may also be delivered to a receiver where each analyte might be stored in separate containers in a manner known as fraction collection. In some cases, a small amount of the column effluent may be directed to the inlet of another sample analysis device, such as a mass spectrometer to further analyze each individual analyte. The delivery of at least a portion of the column effluent to a further liquid analysis device is referred to as “second dimension” analysis, and is commonly employed in complex liquid analysis.
An example application for two dimensional liquid analyses is in the purification of a synthesized compound during the development of a new drug. Often, the products of the synthesis include the desired synthesized compound (with a known molecular weight), reactants and side products, all of which are analytes in the synthesis sample. In this example, a “first dimension” analysis carries out analytical or preparative scale separation, such as through an HPLC column, with a dedicated detection means such as a high flow rate refractive index detector or an ultraviolet light detector monitoring column effluent. A “second dimension” analysis may preferably utilize a second, separate flow path to capture a portion of the column effluent and direct the flow to a secondary analysis device, such as a mass spectrometer. Such combined instruments in a “two-dimensional” arrangement are becoming increasingly used to extend the understanding of the purity of compounds in a liquid scale.
For a second-dimension analysis device, such as a mass spectrometer, to function optimally, a controlled low mass rate of the eluent from the first dimension HPLC column containing the analyte should be delivered. Such mass or flow rates should be easily adjustable and closely controllable despite variations in the flow rate of the first dimension system. The flow rate should be reproducibly controlled, which facilitates second-dimension identification of the purity of an eluting peak of the desired synthesized compound to allow the collection of pure analyte in individual fractions. An experienced analyst may select a desired carrier fluid to transfer the analyte into the second-dimension detector, which second dimension carrier fluid may be different from the mobile phase used to perform the first-dimension preparative separation of the synthesized compound. Certain mobile phase fluids used to perform chromatographic separations may contain dissolved buffer salts which can cause fouling of a different second dimension analysis device such as a mass spectrometer, and certain organic components of the mobile phase can inhibit optimum ionization of the analytes which is required in a mass spectrometer. Proper selection of the carrier solvent reduces the effect on the mass spectrometer of the first-dimension analyte-mobile phase being transferred into the mass spectrometer. In addition, the analyte mass transfer rate into the mass spectrometer should be small, and generally should be a small fraction of the total analyte flow rate in the first dimension. A large mass rate to a mass spectrometer can result in a lingering or tailing signal that distorts the results of a mass spectrometer, and a large mass rate can change the dielectric properties of the system and cause a momentary loss of signal.
Some forms of secondary analysis devices may be better suited for receiving inlet flow at a rate that is significantly less than the flow rate typically passed through an HPLC separation column. Although modern mass spectrometers are designed with sample introduction systems wherein the flow rate of the inlet mobile phase can be as much as several milliliters per minute, such mass spectrometers utilize expensive high volume turbo molecular pumps and high volume roughing pumps to handle the large solvent loads. Reducing inlet flow rate can reduce or eliminate the need for such expensive equipment, and may also facilitate superior second dimension analysis. A desired approach, therefore, for second dimension liquid analysis is to supply only a representative portion of the first dimension flow to the second dimension analysis device. An example conventional mechanism for diverting a small fractional volume of analytes from a first dimension analysis system is shown in U.S. Pat. Nos. 6,890,489, and 7,575,723, assigned to the same assignee as in this application, and incorporated herein by reference.
Conventional “mass rate attenuators” or flow diversion apparatus typically do not permit sustained, continuous flow of a secondary analysis stream to a second dimension analysis device, which may be harmful to sensitive analysis equipment, such as in mass spectrometers. Moreover, conventional devices employ a “back and forth” switching mechanism transferring the analyte from the first dimension analysis to the secondary flow stream for the second dimension analysis, and then returning the carrier fluid flowing in the secondary flow stream back into the first dimension effluent. Returning the second dimension carrier liquid to the first dimension flow can contaminate the first dimension flow, which can frustrate efforts to obtain separated and purified analytes in the first dimension effluent.
It would therefore be of value to provide a device that is capable of separating out a very small closely controlled portion of a larger first dimension stream, and divert that portion along a secondary path without returning any portion of the flow from the second dimension to the first dimension flow stream.